SK492002A3 - Hydroxylation of compactin to pravastatin by micromonospora - Google Patents

Abstract

The present invention relates to a new microbial process for the preparation of compound of formula (I) from a compound of general formula (II) wherein R stands for an alkali metal or ammonium ion, by the submerged culture of a strain which is able to 6 beta -hydroxylate the compound of formula (II) in aerobic fermentation and by the separation and purification of the product of formula (I) formed in the course of the bioconversion. The latter comprises the cultivation of a Micromonospora strain which is able to 6 beta -hydroxylate a compound of general formula (II) - wherein R is as defined above - at 25-32 DEG C on a nutrient medium containing available carbon - and nitrogen sources and mineral salts, thereafter feeding the substrate to be transformed into the developing culture, then hydroxilating the substrate until finishing of the bioconversion, then separating the compound of formula (I) from the culture broth and, if desired, purifying the same.

Description

Technical field

The present invention relates to a novel microbial process for preparing pravastatin.

More specifically, the present invention relates to a microbial process for preparing pravastatin of formula I

wherein R is an alkali metal or ammonium ion, with a microorganism, said microorganism being a prokaryote of the genus Micromonospora, which is capable of hydroxyl of a compound of formula II at the 6β position.

BACKGROUND OF THE INVENTION

Hypercholesterolaemia is considered to be a major risk factor in atherosclerotic disease, especially coronary heart disease. Over the past two decades, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase EC. 1.1.1.34) has been extensively investigated as a major rate-limiting enzyme in cholesterol synthesis. Mevinoline and related compounds have been found to be biologically synthesized using selected strains of different fungal species to be competitive inhibitors of this enzyme [Endo, A. et al., J. Antibiotics 29, 1346 1348 (1976); Endo, A. et al., FEBS Lett. 72, 323-326 (1976); Kuo, C. H. et al., J. Org. Chem. 48, 1991-1998 (1983)].

Pravastatin is also a member of the HMG-CoA reductase inhibitor family. Pravastatin was first found as a minor metabolite of compactin in dog urine (Tanaka, M. et al., Unpublished results) during compactin metabolic studies [Arai, M. et al. Sankyo Kenkyusho Nempo, 40, 1-38 (1988)].

The major characteristic of pravastatin as a hydroxylated compactin product is its tissue selectivity. This drug strongly inhibits sterol synthesis in the liver and small intestine, but weakly in other organs. It is preferred that pravastatin exhibits lower toxicity than other HMG-CoA reductase inhibitors.

It has been reported that microbial hydroxylation of compactin can be accomplished to varying degrees using certain strains belonging to several diverse fungal genera and using strains of actinomycetes belonging to the genera Nocardia, Actinomadura and Streptomyces, among others Streptomyces roseochromogenes and Streptomyces carbides US Patent Documents US 5,179,013, US 4,448,979, US 4,346,227, US 4,537,859, Japanese Patent Document No. 58,010,572).

The problem with using fungi to produce pravastatin from compactin is that these organisms are generally not tolerant of higher concentrations of compactin in the liquid culture medium, probably because of their fungicidal activity [Serizawa, N. et al., J. Antibiotics 36, 887-891 (1983)]. Streptomyces carbophilus has been shown to require a cytochrome P450 system when hydroxylating compactin to pravastatin [Matsuoka, T. et al., Eur. J.

-3Biochem. 184, 707-713 (1989)], The difficulty of genetically improving the ability to hydroxylate using such an enzyme is that it is a protein complex rather than a single protein.

SUMMARY OF THE INVENTION

Our investigations have focused on finding an actinomycetes strain that produces pravastatin from salts of the acid form compactin with higher yields and using higher substrate concentrations in biological conversion than is known from prior patents.

During screening, which included approximately 6000 actinomycetes, mostly our own isolates, but also native strains from international collections, five Streptomyces and five Micromonospora were chosen for further studies because they proved to be able to hydroxylate the sodium form of compactin to pravastatin. The ten actinomycetes strains, eight of which were taxonomically identified at the species level in our laboratory, were as follows:

Streptomyces violaceus (according to Kämpfer et al., 1991), strain no. 1/43.

Streptomyces rochei (Berger et al., 1949; Waksman and Lechevalier, 1953), strain no. 1/41.

Streptomyces resistomycificus (Lindenbein, 1952), strain no. 1/44.

Streptomyces sp., Strain No: 1/28.

Streptomyces lanatus (Frommer, 1959), strain no. 1/16.

Micromonospora sp., Strain no. IDR-P ₃ .

Micromonospora purpurea (Luedemann and Brodsky, 1964), strain no. IDR-P ₄ .

Micromonospora echinospora (Luedemann and Brodsky, 1964), strain no. IDR-P ₅ .

Micromonospora megalomicea (Weinstein et al., 1969), strain no. IDR-P ₆ .

Micromonospora rosaria (Horan and Brodsky, 1986), strain no. IDR-P ₇ .

Since so far there is no data in the literature about the ability of Micromonospora to convert salts of the acid form of compactin to pravastatin, we have carefully studied not only this special enzymatic ability, but also the taxonomic position of these Micromonospora strains.

Generic level of IDR-P ₃ , -P ₄ , -P ₅ , -P ₆ and -P ₇ strains

All of these strains produced well-developed mycelia, consisting of a branched herd with a diameter of about 0.4 to 0.7 µm. Aeric mycelium is absent or occurs only in traces. Still spores were formed on sporophores separately. The mycelium hyphae of the substrate are Gram-positive and are not acid-resistant. Strains no. IDR P3 to _P7 are aerobic, chemoorganotrofické and sensitive to pH below 6.0. The walls contain mesodiaminopimelic acid. The above diagnostic properties - as key characteristics - clearly demonstrate that these monosporic actinomycetes strains are typical members of the genus Micromonospora.

Taxonomic description of Micromonospora sp. IDR-P ₃

Micromorphological properties: The mycelium of the substrate consists of well developed, more curved than straight, monopodially branched fibers. Spores on sporophores are simple, spherical with a diameter of approximately 1.8 μπι, and dispersed more or less on the fibers of the vulture. The spores are either attached (without the stem) or at the end of short sporophores. In the mother cultures, spores were not observed on the hatch, probably because the release of mature spores is very rapid.

Culture-macromorphological properties:

Czapek's sucrose agar: Growth of the medium, colonies are reddish in color covered with dotted black sporulation areas.

Glucose-asparagine agar: Growth was recorded as pointed and elevated, reddish-brown or black colonies. Reddish intertwined pigments.

Nutrient agar: Average growth, elevated, reddish-brown or black colonies. Reddish exopigment in medium.

Yeast Extract - Malt Extractgar (ISP Med.2): Well developed, elevated and wrinkled brown colonies, partially covered with black sporulation areas or with pseudo-aerial mycelium '(this appears as a bordered whitish or greyish block). Brownish or brownish-red soluble pigment.

Inorganic Salt Starch Agar (ISP Med. 4): Growth of reddish-brown raised and wrinkled colonies. Light reddish soluble pigment.

Glycerol-asparagine agar (ISP Med. 5): Growth in traces only, not completely white or light orange color, flat colonies, light pink soluble pigment.

Carbon source utilization: Good growth for and positive utilization of Larabinose, D-galactose, D-fructose, D-glucose, D-xylose, lactose, melibiosis, sucrose, D-mannitol, dulcitol, glycerol and inositol. Growth with L-rhamnose, Drafinose and inulin was slightly better than on the negative control medium.

Utilization of nitrogen source: Growth with yeast extract and NZ-amine, no or poor availability of NaNO ₃ .

Other physiological-biochemical properties: Cellulose and starch were hydrolyzed, the milk decomposed strongly. The nitrogen reduction test was negative. No growth was observed on calcium carbonate-free potato shreds (pH 5.8-6.0) No production of melanoid pigment was observed.

This strain no. IDR-P ₃ of Micromonospora was isolated from a sludge sample from Lake Balaton (Hungary).

Systematic classification: Further comparative studies would be needed to clarify the exact taxonomic classification of this strain among Micromonospora species. Due to certain properties, it seems not impossible that the strain IDR-P ₃ may represent new species within the genus Micromonospora.

Differential diagnostic description and identification of Micromonospora IDR-P4, -P5, -Ρβ and -P7 strains

-6-IDR-P4 strain

On the above diagnostic media, generally, good growth, orange to orange-red, red, sometimes lived or pink colored colonies. Soluble pigments and aerial mycelium were not produced. The number of individual spores is relatively low. They occur terminally on sporophores. The mycelium of the substrate consists of well branched hyphae. Aeric mycelium is missing. No growth on D-melibiosis, raffinose, mannitol, glycerol, lactose, L-rhamnose, but good growth on D-arabinose, glucose, D-xylose and poor growth on D-galactose and D-fructose. Based on these conventional diagnostic properties, we have identified this strain as a member of Micromonospora purpurea (Luedemann and Brodsky, 1964).

IDR strain P5

This strain produces predominantly separate sporophores and spherical dark brown to black spores (0.8-1.5 µm in diameter) that adhere firmly to the sporophores until maturation. According to our observations using an electron microscope, on the surface of these spores we can see wart structures or growths ("blunt spines" according to Vol. 4 of the Bergey's Manual of Syst. Bact. 1989, p. 2448), which is very characteristic for spores of Micromonospora echinospora. Otherwise, the culture-morphological and physiological diagnostic properties of this strain are also very similar to those of M. echinospora. The color of well developed colonies on standard diagnostic media is orange-brown or dark purple. The sporulating layer is black or magenta-black, waxy. Aeric mycelium is missing. Melanin pigment is not formed. Milk decomposes. Good growth on D-xylose, D-arabinose, D-glucose and sucrose but no growth with L-rhamnose, raffinose, D-galactose, D-fructose, D-melibiosis and glycerol. We believe that this strain is a typical member of Micromonospora echinospora.

IDR-P strain ₆

Medium to weak growth on most diagnostic media. Orange or orange-red colonies consisted of long branched fibers (approximately 0.6 µm in diameter) and a limited number of individual, spherical, dark colored

-7 spores (0.6-1.0 µm in diameter). It does not produce an aerial mycelium, weakly reddish or pink colored soluble pigments have formed in certain media. Melanoid pigments were not produced on tyrosine agar. The following carbon sources were used on the base medium: D-xylose and D-fructose; only slightly: D-melibiosis, mannitol and galactose but no or sporadic growth was observed with glycerol, L-rhamnose, lactose and raffinose (see also Kawamoto, I. et al., Agric. Biol. Chem., 47, 203- 215 (1983). Tribe no. IDR-P6 showed considerable similarity to Micromonospora megalomicea, (Weinstein, 1972) and is considered a member of that species.

IDR-Py strain

Good or moderate growth on Bennett agar, Czapek sucrose agar, glucose-asparagine agar, nutrient agar, oatmeal agar, potato-dextrose agar and the like. The color of the vegetative mycelial pigments ranged from reddish-brown to purple-brown. Wine-red diffuse pigments have been formed on certain media. Black spots often formed on the surface of the colonies. Vegetative hyphae (mean diameter 5 µm) branched intensively. Spores (1.4-1.7 µm in diameter) formed alone, ingrown (without stalk) or on short sporophores, and occurred along the length of the veneer. Growth and sporulation were Luedemann's open full-body type. This strain used the following compounds as the sole carbon source in the media: D-glucose, lactose, D-mannitol, Lramnose, sucrose and D-xylose: Dulcitol, glycerol, D-melibiosis and D-raffinose were not used. We identified strain no. IDR-P ₇ as a typical member of Micromonospora rosaria (Horan and Brodsky, 1986).

The above Micromonospora strains were deposited with the National Collection of Agricultural and Industrial Microorganisms (NCAIM), Budapest, Hungary, under the following code numbers:

Micromonospora sp. IDR-P ₃

Micromonospora purpurea IDR - P ₄

NCAIM (P) B 001271

-8Micromonospora echinospora ssp. echinospora IDR P ₅ NCAIM (P) B 001272 Micromonospora megalomicea ssp. nigra IDR-P ₅ NCAIM (P) B 001273

Micromonospora rosaria IDR-γ NCAIM (P) B 001274

Based on the above, the present invention relates to a novel microbial process for preparing pravastatin of formula I

O) from a compound of formula II

OD wherein R is an alkali metal or ammonium ion by submerged strain cultivation capable of 6β-hydroxylation of a compound of Formula II in aerobic fermentation and separation and purification of a compound of Formula I formed during biological conversion comprising the steps of

(a) culturing a strain of a species belonging to the genus Micromonospora which is capable of beta-hydroxylation of a compound of formula II - wherein R is as defined above - for

- 9 a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts at a temperature of 25 to 32 ° C, followed by

b) feeding the substrate to be transformed into the developed culture then

c) hydroxylating the substrate until the biological conversion is complete, then

d) separating the compound of formula I from the culture broth and, if necessary, purifying it.

Included within the scope of the present invention are "wild strains" and all mutants of species belonging to the genus Micromonospora that are capable of hydroxylating the sodium salt of compactin acid to pravastatin.

According to a preferred embodiment of the present invention pravastatin is produced with a Micromonospora strain selected from the group consisting of Micromonospora IDR-P ₃ [NCAIM (P) B 001268], Micromonospora purpurea IDR-P ₄ [NCAIM (P) B 001271], Micromonospora echinospora IDR-P5 [NCAIM (P) B001272], Micromonospora megalomicea IDR P ₆ [NCAIM (P) B 001273], and Micromonospora rosaria IDR-P ₇ [NCAIM (P) B 001274],

According to a most preferred embodiment of the present invention, pravastatin is produced by a strain of Micromonospora sp. IDR-P ₃ [NCAIM (P) B 001268],

The process of the present invention can be carried out by an in situ fermentation method, that is, the hydroxylation is carried out with an actively growing Micromonospora culture.

Hydroxylation can be accomplished by shaking the culture in a shake flask or by aerating and shaking in fermenters after the compound of formula I1 has been added to the growing cultures. In such cases, an antifoaming agent may be used. An adequate culture density of this strain can be achieved by using a suitable medium containing useful carbon and nitrogen sources; inorganic salts as well as trace elements.

For example, glucose, glycerol, dextrin, starch, rhamnose, xylose, sucrose and soluble starch have proven to be assimilatable carbon sources, while soybean

-10meal, corn extract, peptone, yeast extract, meat extract, ammonium citrate and ammonium sulfate have proven to be good sources of nitrogen. Inorganic salts such as calcium carbonate, sodium phosphate, potassium phosphate and the like can be added to the culture medium. Preferred media for growth of this selected strain are those described in the Examples.

Biological conversion of compactin to pravastatin can be accomplished using a variety of fermentation methods, such as batch culture, batch culture. Preferably, a shaken liquid submerged culture is used. A preferred temperature is about 25 ° C to 37 ° C, most preferably about 25 ° C to 32 ° C.

The preferred pH is about 6.0 to 9.0, particularly preferably about 7.0 to 8.5. Preferred shaking conditions are about 200 rpm to 400 rpm, most preferably about 250 rpm.

The present invention provides a process for converting compactin acid sodium salt to pravastatin. The compactin acid sodium salt of the present invention can be used at any concentration that provides pravastatin production. Preferably, the concentration of compactin is between 0.1 and 10 g / liter, particularly preferably between about 0.3 and 3.0 g / liter.

The present invention is to be understood to include any percent conversion of compactin to pravastatin using strains of Micromonospora spp., At least 30%, and particularly preferably at least about 90%.

During the fermentation, the culture broth composition is checked by the High Performance Liquid Chromatography (HPLC) method. According to the HPLC method, a sample of broth is diluted twice with methanol, centrifuged and the supernatant used for further analysis. The HPLC system parameters to be used for the analysis are: Waters Analytical HPLC; column packing: Waters Novapack C ₁₈ 5 μιτι; measurement at 237 nm; injection volume 10 μΙ; a flow rate of 0.6 to 0.9 ml / min linear gradient; gradient elution was used, eluents: solvent A = acetonitrile - 0.1 M NaH ₂ PO ₄ in water (25: 75), solvent B = acetonitrile - water (pH 2 with H ₃ PO ₄ ) (70:30 ratio) ).

-11 Gradient elution parameters:

Time (min) Flow rate (ml / min) Eluent A (%) Elution Reagent B (%) 0 0.6 100 0 2 0.7 100 0 12 0.9 0 100 21 0.9 0 100 22 0.9 100 0 27 0.7 100 0

Retention times: pravastatin (Na-salt) 10.6 min; compactin (acid Na-salt)

19.5 min; pravastatin (lactone form) 17.3 min, compactin (lactone form)

23.5 min.

Any known procedure may be used to isolate pravastatin, for example extraction-reextraction, anion exchange chromatography, precipitation.

In order to recover the broth product, it is advantageous to take into account the fact that pravastatin is produced in acid form during the biological conversion, i.e. the broth filtrate can be isolated by adsorption on an anion exchange resin column. It is preferred that a strongly basic anionic ion exchange resin, which is a polystyrene-divinylbenzene polymer carrying quaternary ammonium active groups, is used, for example, Dowex Al 400 (OH '), Dowex 1 x 2 (OH'), Dowex 2x4 (OH) Amberlite IRA 900 (OH). The product adsorbed on the ion exchange resin can be eluted from the column using aqueous acetic acid or sodium chloride containing acetone-water, preferably using 1% sodium chloride containing acetone-water (1: 1 ratio). The fractions containing pravastatin are combined and the acetone in the eluate is distilled off in vacuo. The pH of the concentrate is adjusted with 15% sulfuric acid to a range of 3.5 to 4.0 and the acidified aqueous solution is extracted using ethyl acetate. Pravastatin can be extracted from the ethyl acetate extract using a 1/10 and 1/20 volume ratio of 5% sodium bicarbonate or weakly alkaline water (pH

7.5 to 8.0). It has been found that pravastatin can be obtained in pure form from above

-12 of the alkaline aqueous extract obtained by column chromatography on a non-ionic adsorption resin. Preferably, the ethyl acetate dissolved in the aqueous phase is first removed from the alkaline aqueous extract by vacuum distillation, and then the aqueous extract is applied to a Diaion HP-20 column. The pravastatin adsorbed on the column is purified by elution with aqueous acetone in which the acetone content is gradually increased, then the chromatographic fractions containing pravastatin as the only component are combined and concentrated in vacuo. The concentrate is purified with charcoal and lyophilized, then crystallized from ethanol-ethyl acetate to give pravastatin in a quality acceptable for pharmaceutical use.

After completion of the biological conversion, pravastatin can be extracted either from the fermentation broth or from the filtrate obtained after separation of the mycelium mass. The latter can be removed either by filtration or by centrifugation, but it is advantageous, especially on an industrial scale, to extract the whole broth. Prior to extraction, the pH of either the fermentation broth or the broth filtrate is adjusted to 3.5-3.7 with a mineral acid, preferably dilute sulfuric acid. The extraction is carried out with an acetic acid ester of 2 to 4 carbon atoms containing an aliphatic alcohol, preferably ethyl acetate or isobutyl acetate. The ethyl acetate extract was washed with water and dried over anhydrous sodium sulfate. A lactone derivative is then prepared from pravastatin. The lactone ring closure is carried out in a dried solution of ethyl acetate at room temperature, with continuous stirring, inducing the formation of lactone with a catalytic amount of trifluoroacetic acid. Lactone ring closure was verified by thin layer chromatography (TLC) analysis. After completion of the lactone formation, the ethyl acetate solution is washed first with 5% aqueous sodium hydrogen carbonate solution and then with water and evaporated in vacuo. The mixture was purified by silica gel column chromatography using ethyl acetate-n-hexane as eluent, gradually increasing the ethyl acetate content. Pravastatin is prepared from pravastatin lactone by hydrolysis at room temperature in acetone with an equivalent amount of sodium hydroxide. Upon completion of pravastatin sodium formation, pravastatin precipitates with acetone. The precipitate is then filtered off and washed with acetone and n-hexane and dried in vacuo, then crystallized from ethanol-ethyl acetate.

It has been found that Sephadex LH-20 gel chromatography can be advantageously used to purify pravastatin. With this method pravastatin can be prepared with an excellent purity of 99.5% (measured by HPLC).

In the course of our experiments, the following findings have been found: from an organic solvent extract, preferably from ethyl acetate extract or isobutyl acetate extract of broth or broth filtrate of the strain Micromonospora sp. IDR-β ₃ , which is capable of 6β-hydroxylation of a compound of formula II, pravastatin may precipitate as a crystalline salt with secondary amines. In addition, some secondary amines containing alkyl, cycloalkyl, aralkyl or aryl substituents have been found suitable for salt formation. Among these, suitable non-toxic secondary amines, such as dioctylamine, dicyclohexylamine, dibenzylamine, have been selected. Isolation of organic secondary amine salt intermediates such as dibenzylamine salt was accomplished by adding 1.5 equivalents of dibenzylamine based on the pravastatin content of the extract, then concentrating the extract by vacuum distillation to 5% of its original volume, followed by additional dibenzylamine in the concentrate. ratio of 0.2 equiv. The crystalline dibenzylamine salt precipitated from the concentrate. The crystalline crude product was filtered and dried under vacuum and purified with activated carbon in methanol or acetone solution. Thereafter, a chromatographically pure intermediate of pravastatin dibenzylamine salt can be obtained by recrystallization of the purified product from acetone.

The organic secondary amine salts of pravastatin can be transformed into pravastatin using sodium hydroxide or sodium alkoxide, preferably sodium ethoxide.

Isolation of pravastatin by the intermediate amine salt intermediate is a simpler procedure than any of the prior art isolation procedures. Artifacts are not formed during the process and the separation of pravastatin from the by-products of biological conversion and from the various metabolic products biologically synthesized by hydroxylation of the microorganism can advantageously be solved.

The method of the present invention is illustrated by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Spores were obtained from the surface of a 7-10 day slant culture of Micromonospora sp. IDR-P ₃ [NCAIM (P) B 001268 soluble starch agar (SM) and suspended in 5 ml of sterile distilled water. This suspension was then used to inoculate 100 ml of sterile TI inoculation medium in a 500 ml Erlenmeyer flask.

Composition of SM media

Soluble starch 10.0 g

Na ₂ HPO ₄ 1.15 g

KH ₂ PO ₄ 0.25 g

KCl 0.2 g

MgSO ₄ x 7 H ₂ O 0.2 g

Agar 15.0 g in 1000 ml of distilled water

The pH of the medium was adjusted to 7.0 prior to sterilization and the mixture was sterilized at 121 ° C for 25 minutes.

TI media composition

Soluble starch 20.0 g

Yeast extract 10.0 g in 1000 ml tap water

The pH was adjusted to 7.0 before sterilization and heat-treated at 121 ° C for 25 minutes.

The evolving culture was shaken on a rotary shaker (250 rpm; amplitude:

2.5 cm) for 3 days at 32 [deg.] C., then a 5 ml aliquot was used on

-15 inoculate ten 500-ml Erlenmeyer flasks each containing 100 ml of TT medium sterilized at 121 ° C for 25 minutes.

Composition of TT media

Potato starch 30.0 g

Soya meal 30.0 g

CaCC> 3 5.0 g

CO 2 x 6 H 2 O 2.0 mg

Palm oil 2.0 g in 1000 ml tap water

The pH was adjusted to 7.0 before heat sterilization.

Incubation was performed at 32 ° C for 72 hours, then 50 mg of compactin acid sodium salt in distilled water was added to each flask and cultivation was continued for 96 hours. The conversion rate of compactin acid sodium salt to pravastatin as measured by HPLC was 82%.

After fermentation, the cultures were pooled and recovered from the common fermentation broth containing 410 mg pravastatin as follows: The fermentation broth was centrifuged at 2500 rpm for 20 minutes. The broth supernatant and the mycelial mass were separated, then the latter was resuspended in 250 ml of water and the resulting suspension was stirred for one hour and then filtered. The pH of the combined centrifuged broth and filtrate was adjusted to 4.0 using 15% sulfuric acid, then the acidic filtrate was extracted with 3 x 300 mL ethyl acetate. The combined ethyl acetate extracts were washed with 300 mL of water, dried over anhydrous sodium sulfate and concentrated in vacuo to a volume of 100 mL. Thereafter, pravastatin lactone was prepared from pravastatin by adding trifluoroacetic acid in a catalytic amount at room temperature with continuous stirring. Pravastatin lactone formation was monitored by TLC method: adsorbent: Kieselgel 60 F254 DC (Merck) aluminum foil; developing solution: acetone-benzene-acetic acid (50: 50: 1.5); detection: with phosphomolybdenoic acid reagent. Pravastatin lactone Rf value

- 16 was 0.7. After completion of the lactone formation, the ethyl acetate was washed with 2 x 20 mL of 5% aqueous sodium bicarbonate solution, then washed with 20 mL of water, dried over anhydrous sodium sulfate and evaporated in vacuo. 0.5 g of evaporated residue was obtained, which was chromatographed on a column containing 10 g of Kieselgel 60 adsorbent (column diameter: 1.2 cm, adsorbent bed height: 17 cm). Ethyl acetate-n-hexane was used to elute and the ethyl acetate content gradually increased. Pravastatin lactone was eluted from the column with 60% ethyl acetate-40% n-hexane. The pravastatin lactone containing fractions were combined and evaporated in vacuo. The residue, which contained 230 mg pravastatin lactone, was dissolved in 5 ml acetone and then 110 mol% sodium hydroxide in 1 M ethanol solution was added with stirring. Stirring of the solution was continued for half an hour at room temperature. The solution was then concentrated to 2 ml and 4 ml of acetone was added to the concentrate. The mixture was kept at + 5 ° C overnight. The precipitate was filtered off, washed with 2 ml of acetone and then with 2 ml of n-hexane and dried in vacuo at room temperature. The resulting crude pravastatin was dissolved in ethanol, purified with charcoal, then crystallized from ethanol-ethyl acetate. 170 mg of pravastatin were obtained.

Melting point 170-173 ° C (decomposition) [α] ^D = + 156 ⁰ (c = 0.5, in water).

Ultraviolet absorption spectrum (20 µg / ml, in methanol): λ max = 231, 237, 245 nm (log ε = 4.263; 4.311; 4.136).

Infrared Absorption Spectrum (KBr): vOH 3415, v CH 2965, v C = 0 1730, v COO 1575 cm ^-1 .

¹ H-NMR spectrum (D ₂ O, δ, ppm): 0.86, d, 3H (2-CH ₃ ); 5.92, dd, J = 10.0 and 5.4 Hz, 1H (3-H): 5.99, d, J = 10.0 Hz, 1H (4-H); 5.52, br. 1H (5-H); 4.24, m, 1 H (6-H); 5.34, br. 1H (8-H); 4.06, m, 1H (β-Η), 3.65, m, 1H (δ-Η); 1.05, d, 3H (2'-CH _3); 0.82, t, 3H (4'-H ₃ ).

-17 ¹³ C-NMR (D ₂ 0, δ, ppm): 15.3, q (2-CH _3,); 139.5, d (C-3); 129.5, d (C-4); 138.1, s (C-4a); 127.7, d (C-5); 66.6, d (C-6); 70.1, d (C-8); 182.6, s (COO '); 72.6, d (C-b); 73.0, d (C-δ); 182.0, s (C-1 '); 18.8, q (2'-CH _3); 13.7, q (C-4 ').

Positive FAB mass spectrum (characteristic ions): [M + Na] + 469; [M + H] + 447. Negative FAB mass spectrum [characteristic ions]: [M-H] - 445, [MNa] - 423, m / z 101 [2-methyl-butanoic acid -H] -.

Example 2

Ten 500 ml Erlenmeyer flasks, each containing 100 ml MTi bioconversion medium, were seeded with an inoculation culture prepared as described in Example 1, then incubated at 28 ° C for 96 hours and 50 mg added to each flask. compactin sodium salt in distilled water, hydroxylation was then performed for 72 hours, when an additional 50-50 mg of substrate in distilled water was added to the cultures and the fermentation was continued for 72 hours.

Composition of bioconversion medium MT ₁

Potato starch 10.0 g

Dextrose 20.0 g

Soya meal 10.0 g

Yeast extract 10.0 g

CaCO3 5.0 g

Sunflower oil 2.0 g in 1000 ml tap water

The pH of the bioconversion medium was adjusted to 7.0 before sterilization. The mixture was sterilized at 121 ° C for 25 minutes.

At the end of the biological conversion period, the cultures were pooled and pravastatin was isolated from the total broth in accordance with the following procedure:

The pooled broth containing 750 mg pravastatin, as determined by HPLC, was centrifuged at 2500 rpm for 20 minutes. Separated matter

The 18 mycelium was stirred with 250 ml of water for one hour, then filtered. The centrifuged broth and filtrate were combined and the pH of the resulting solution was adjusted to 3.5-4.0 with 15% sulfuric acid, then the solution was extracted with 3 x 300 mL ethyl acetate. Then, 150 mole percent of dibenzylamine - based on pravastatin content - was added to the ethyl acetate extract. The ethyl acetate extract was evaporated to approximately 30 mL volume and the suspension was kept overnight at 0-5 ° C. The precipitated acid dibenzylamine salt of pravastatin was filtered off and washed on a filter with cold ethyl acetate and β-hexane, finally dried in vacuo. 1.1 g of the crude acid dibenzylamine salt of pravastatin was dissolved in 33 ml of acetone at 62-66 ° C and the solution was purified with 0.1 g of activated carbon for half an hour. Subsequently, the activated carbon was removed from the solution by filtration. The crystals precipitated from the purified solution were redissolved at the above temperature, then the solution was kept overnight at + 5 ° C. The precipitate was filtered off, washed with cold acetone and n-hexane and dried in vacuo. The obtained pravastatin acid dibenzylamine salt (0.7 g) was suspended in 10 ml of ethanol, then 110 mol% sodium hydroxide was added as a 1 M aqueous solution. Stirring of the alkaline solution was continued for half an hour at room temperature. After the formation of the sodium salt was complete, 30 ml of water was added and the pH of the solution was neutralized, and then the ethanol was distilled off in vacuo. The aqueous concentrate was chromatographed on a column packed with 50 ml of Diaion HP 20 resin (column diameter: 1.5 cm, resin bed height 28 cm). The column was eluted with acetone-deionized water, increasing the acetone concentration in 5% steps. Pravastatin was eluted from the column with 15% acetone containing acetone-deionized water. Fractions were analyzed using the TLC method of Example 1. The pravastatin R _f value was 0.5. The fractions containing pravastatin were combined and the acetone content was evaporated in vacuo. Lyophilization of the aqueous residue gave 390 mg of chromatographically pure pravastatin.

Example 3

4.5 liters of TT / L medium was sterilized in a laboratory fermenter at 121 ° C for 45 minutes and then inoculated with 500 ml. shake culture prepared as described in Example 1, then incubated at 32 ° C, aerated with 250 L of sterile air / hour and mixed

-19 using a flat blade stirrer at 300 rpm. Incubation was continued for 72 hours and 2.5 g of compactin acid sodium salt was added to the culture. After a 48 hour biological conversion period, the compactin substrate was completely consumed from the fermentation broth, then an additional 2.5 g of compactin acid sodium salt was added to the culture. The second dose of compactin was consumed within 24 hours. The conversion rate of compactin acid sodium salt to pravastatin was about 90% in the biological conversion process.

Composition of TT / 2 bioconversion medium

Glucose 75.0 g

Soluble starch 50.0 g

Soya meal 50.0 g

Yeast extract 50.0 g

Peptone 5.0 g

NaNO ₃ 20.0 g

CaCO ₃ 25.0 g in 4500 ml tap water

Example 4

4.5 liters of TT / L fermentation broth were sterilized in a laboratory fermentor at 121 ° C for 45 minutes and then inoculated with 500 ml. shake culture prepared as described in Example 1, then the mixture was incubated at 28 ° C, aerated with 200 L of sterile air / hour, and mixed using a flat blade stirrer at 400 rpm.

Composition of TT / 1 bioconversion medium

Glucose 125.0 g

Potato starch 25.0 g

Soya meal 50.0 g

Yeast extract (Gistex) 50.0 g

-20Peptón

CoCl ₂ x 6 H ₂ O

Sunflower oil

50,0 g

10.0 mg

10.0 g in 4500 ml of tap water

The pH of the bioconversion medium was adjusted to 7.0 before sterilization.

Cultivation was continued at 28 ° C for 96 hours. Then 2.5 g of compactin acid sodium salt in a sterile filtered aqueous culture solution were added. On the fifth day of fermentation, the compactin acid sodium salt was completely consumed from the fermentation broth. Substrate dosing was then repeated daily for a further 3 days at doses of 2.5 g / day. The compactin acid sodium salt substrate was gradually consumed over four days and completely converted to pravastatin. According to HPLC measurements at the end of the fermentation period, 9 g pravastatin was produced from 10 g compactin substrate.

After completion of the biological conversion, pravastatin prepared at a concentration of 1800 pg / ml was isolated as follows:

L of culture broth was centrifuged at 2500 rpm for 20 minutes. Then 2 liters of water were added to the separated mycelial meat and the suspension was stirred for one hour and filtered. The two filtrates were combined and passed at a flow rate of 500 ml / hour through a column containing 300 g (540 ml) of Dowex A1 400 (OH ') resin (column diameter: 4 cm, resin bed height: 43 cm), then washed with 1 liter of deionized water. Subsequently, the column was eluted with 1 liter of acetone-water (1: 1) containing 10 g of sodium chloride, collecting 50 ml of fractions. Fractions were analyzed using the TLC method of Example 1. Fractions containing the product were combined and acetone distilled off in vacuo. The pH of the concentrate was adjusted to 3.5-4.0 using 15% sulfuric acid, then the concentrate was extracted with 3 x 250 mL ethyl acetate. To the combined ethyl acetate extract was added 40 ml. of deionized water, the pH was adjusted to 7.5 to 8.0 using 1 M sodium hydroxide. After stirring for 15 minutes, the aqueous and ethyl acetate phases were separated, the ethyl acetate solution was extracted with 2 x 40 mL deionized water, such as

-21 above. The combined alkaline aqueous solution was then concentrated to a volume of 50 ml and chromatographed on a column packed with 600 ml of Diaion HP2O (Mitsubishi Co., Japan), a nonionic adsorption resin (column diameter: 3.8 cm, resin bed height: 53 cm). The column was washed with 600 ml deionized water, then eluted with acetone-deionized water where the acetone concentration was increased in 5% steps to collect 50 ml fractions. The eluate was analyzed using the TLC method of Example 1. Pravastatin was eluted from the column using acetone-deionized water containing 15% acetone. Fractions containing pravastatin as a single component were combined and the solution concentrated in vacuo to a volume of 150 mL. Subsequently, 0.6 g of activated carbon was added to the concentrated aqueous solution, and pravastatin was purified at room temperature for one hour. The activated carbon was then filtered off and the filtrate was lyophilized. The resulting 6.5 g of lyophilized pravastatin was crystallized twice from a mixture of ethanol and ethyl acetate. The precipitate was filtered off and washed with 20 ml of ethyl acetate and 20 ml of n-hexane and dried in vacuo at room temperature. 4.6 g of chromatographically pure pravastatin are thus obtained.

Example 5

A spore suspension was prepared with 5 ml of sterile distilled water from the surface of a 10-day slant culture of soluble starch agar as described in Example 1, Micromonospora echinospora ssp. echinospora IDR-P ₅ [NCAIM (P) B 001272] - which was capable of 6β-hydroxylation of compactin acid sodium salt - and the obtained spore suspension was used to inoculate 100 µl! inoculation medium T1 sterilized in a 500 ml Erlenmeyer flask. The composition of the T1 medium was as described in Example 1. The inoculated medium was shaken on a rotary shaker (250 rpm, shaking 2.5 cm; for 3 days at 28 ° C, then aliquots of 5 ml of the developed culture were transferred to 100-100 ml of TT / 1 bioconversion medium sterilized in 500 ml Erlenmeyer flasks for 25 minutes at 121 ° C. The composition of TT / 1 medium is described in Example 4. The flasks were shaken on a rotary shaker (250 rpm; swing at 2.5 cm) for 3 minutes. days at 25 ° C, then 10-10 mg of compactin substrate (compactin acid sodium salt) in sterile filtered aqueous solution was added to the cultures and the fermentation was continued for 168 hours.

At the end of the biological conversion, the pravastatin content of the fermentation broth was determined using an HPLC method. The mean pravastatin concentration was 40 pg / ml.

Example 6

Fermentation, substrate dosing and biological conversion were performed with strain IDR-P ₆₁ [NCAIM (P) B 001273] Micromonospora megalomicea ssp. nigra as described in Example 5. The pravastatin content of the fermentation broth was determined using HPLC. At the end of the biological conversion, the pravastatin content in the medium was 50 pg / ml.

Example 7 ml aliquots of IDR-P4 strain inoculation culture [NCAIM (P) B 001271] Micromonospora purpurea, prepared as described in Example 1, was used to inoculate 100-100 ml TT / 14 medium distributed in 500 ml Erlenmeyer flasks and sterilized. at 25 ° C for 25 minutes.

TT / 14 media composition

Potato starch 5.0 g

Glucose 25.0 g

Yeast extract (GISTEX) 15.0 g

Peptone 15.0 g

CaCO3 1.0 g in 1000 ml tap water

The pH of the bioconversion medium was adjusted to 7.0 before sterilization.

The flasks were shaken on a rotary shaker (250 rpm, swing 2.5 cm) for 3 days. Substrate dosing, biological conversion and determination of pravastatin content were performed as described in Example 5. At the end of the biological conversion, the pravastatin content in the fermentation broth was 40 pg / ml.

Example 8

-23Fermentation, substrate dosing and biological conversion were performed with strain IDR-P7, [NCAIM (P) B 001274] Micromonospora rosaria as described in Example 1. At the end of the biological conversion, the fermentation broth was determined to be 350 pg / min in the fermentation broth. ml pravastatin.

Claims (7)

PATENT CLAIMS A microbial process for the preparation of a compound of formula I from a compound of formula II H3C (II) wherein R is an alkali metal or ammonium ion by submerged Micromonospora strain capable of 6β-hydroxylating a compound of Formula II under aerobic conditions and separating and purifying the compound of Formula I formed during biological conversion, comprising action a) culturing a strain of Micromonospora capable of 6β-hydroxylation of a compound of formula II - wherein R is as defined above - at 25 to 32 ° C on a nutrient medium containing useful carbon and nitrogen sources and mineral salts, followed by (b) feeding the substrate to be transformed into the developed culture then -25c) hydroxylating the substrate until the biological conversion is complete, then d) separating the compound of formula I from the culture broth and, if necessary, purifying it. Method according to claim 1, characterized in that the strain Micromonospora sp. IDR-P ₃ deposited with the National Collection of Agricultural and Industrial Microorganisms (NCAIM), Budapest, Hungary, under NCAIM number (P) B 001268, or a mutant strain thereof capable of δβ-hydroxylation of a compound of general of formula II. Method according to claim 1, characterized in that a strain is used Micromonospora purpurea IDR-P ₄ deposited with the National Collection of Agricultural and Industrial Microorganisms (NCAIM), Budapest, Hungary, under NCAIM number (P) B 001271, or a mutant strain thereof capable of 6β-hydroxylation the compound of formula II. Method according to claim 1, characterized in that the strain Micromonospora echinospora ssp. echinospora IDR-P ₅ deposited with the National Collection of Agricultural and Industrial Microorganisms (NCAIM), Budapest, Hungary, under NCAIM number (P) B 001272, or a mutant strain thereof capable of 6β-hydroxylated compound of formula II. Method according to claim 1, characterized in that the strain Micromonospora megalomicea ssp. nigra IDR-Pg deposited with the National Collection of Agricultural and Industrial Microorganisms (NCAIM), Budapest, Hungary, under NCAIM number (P) B 001273, or a mutant strain thereof capable of δβ-hydroxylation of a compound of the general of formula II. Method according to claim 1, characterized in that the strain Micromonospora rosaria IDR-P7 deposited in the National Collection of Agricultural and Industrial Microorganisms (the National Collection) is used. (Agricultural and Industrial Microorganisms, NCAIM), Budapest, Hungary, under NCAIM (P) B 001274 or a mutant strain thereof which is capable of a 6β-hydroxylovaf compound of formula II. Process according to any one of claims 1 to 6, characterized in that the compound of formula I prepared during fermentation is separated from the culture broth by adsorption on an anion exchange resin or by extraction with a water-immiscible organic solvent, followed by preparing its lactone derivative or its secondary the amine salt as an intermediate, or the alkaline aqueous extract obtained from the organic solvent extract of the fermentation broth is purified using chromatography on a nonionic adsorbent resin.